Quantitative real-time polymerase chain reaction, also abbreviated with qPCR, represents a sensitive method for nucleic acid detection and quantification. It is widely used in basic and biomedical research, in vivo diagnostics, and applied testing.
For one or more specific sequences in a DNA sample, quantitative PCR enables both detection and quantification. The quantity can be either an absolute number of copies or a relative amount when normalized to DNA input or additional normalizing genes. The procedure follows the general principle of polymerase chain reaction; its key feature is that unlike in standard PCR, where the PCR product is detected at the end of the PCR, the amplified DNA is detected as the reaction progresses in “real time”.
Generally, two methods for the detection of products in quantitative PCR are employed today, i.e. intercalation of double-stranded DNA-binding dyes on the one hand, and, on the other hand, the use of probes labeled with fluorescent dyes.
Intercalating dyes specific for double-stranded nucleic acids show an increased fluorescence upon intercalating with the double-stranded nucleic acids; as a consequence, the more double-stranded nucleic acid molecules are generated during nucleic acid amplification, the higher gets the increase in fluorescence. Thus, by using intercalating dyes real-time detection of the synthesis of double-stranded PCR products is enabled, and this method allows the initial DNA concentration to be determined with reference to a standard sample. However, and as a consequence, this method also has the drawback that there is no discrimination between correct products and non-specific products such as primer dimers. Also, this method cannot be used to compare levels of different targets. The specificity of the PCR products can be determined by subjecting double-stranded PCR products to incrementally increased temperature to form characteristic dissociation, or melting, curves. With melting curve analysis, the formation of primer dimers and non-specific products can be visualized to monitor qPCR performance.
On the other hand, the second method, i.e. the probe-based detection/Quantification, uses sequence specific DNA-based fluorescence reporter probes, which recognize additional specific sequences within the same PCR amplicon. Sequence specific probes result in quantification of the sequence of interest only and not all ds DNA. The probes contain a fluorescent reporter, such as fluorescein, rhodamine and cyanine, and a quencher to prevent fluorescence. The fluorescent reporter and the quencher are located in close proximity to each other in order for the quencher to prevent fluorescence. Once the probe locates and hybridizes to the complementary target, the reporter and quencher are separated. The means by which they are separated varies depending on the type of probe used. Upon separation, quenching is relieved and a fluorescent signal is generated. The signal is then measured to quantitate the amount of DNA. However, this method, i.e. probe-based qPCR alone, on the other hand, does not give information on whether additional sequences are amplified in the same PCR. Furthermore, in case of suboptimal PCR performance, it is difficult to determine whether PCR failure is caused by inhibitors or conditions leading to formation on non-specific products/primer dimers.
Thus, there still is a need for improved qPCR methods which allow overcoming the above mentioned drawbacks of the methods known in the art.
In view of the above, it is an object of the present invention to provide for tools by means of quantification of a specific target and the total amount of a mixed nucleic acid population can be reliably quantified, and which, in addition, provides for additional advantageous appliances.
The present invention satisfies these and other needs.